CN113728845A

CN113728845A - Method for rooting of Ardisia mamillata Hance tissue culture seedlings outside bottles

Info

Publication number: CN113728845A
Application number: CN202111094005.1A
Authority: CN
Inventors: 邓伦秀; 刘晓慧; 吴高殷; 李鹤; 陈菊艳; 陈志萍
Original assignee: GUIZHOU ACADEMY OF FORESTRY
Current assignee: GUIZHOU ACADEMY OF FORESTRY
Priority date: 2021-09-17
Filing date: 2021-09-17
Publication date: 2021-12-03

Abstract

The invention belongs to the technical field of tissue culture of Ardisia mamillata Hance, and particularly discloses a method for rooting tissue culture seedlings of Ardisia mamillata Hance outside a bottle. According to the matrix and the culture conditions adopted by the method, the pollution rate of the cuttings is almost 0, the cuttings begin to root after 15-20 days of culture, the rooting rate reaches 100% after 25-30 days of culture, the average root length of about 30 days of greenhouse seedbed cutting culture is 3.4cm, the root points are 4.7, the root width is 4-5 cm, the average root length of about 50 days is 6.5cm, the root points are 5.5, and the root width is 6-8 cm, so that seedlings with good root clusters are formed. The invention provides an efficient rooting method for ex-bottle rooting of tissue culture seedlings of Ardisia mamillata Hance, and compared with in-bottle rooting, the efficient rooting method improves the quality of root systems, shortens the rooting time, optimizes the rooting procedure, reduces the production cost, is simple and strong in operability, and can provide technical support for development of the whole industrial chain of Ardisia mamillata Hance.

Description

Method for rooting of Ardisia mamillata Hance tissue culture seedlings outside bottles

Technical Field

The invention belongs to the technical field of tissue culture of ardisia mamillata plants, and particularly discloses a method for ex-vitro rooting of ardisia mamillata tissue culture seedlings.

Background

The red tiger tongue (Ardisia mamillata Hance), also known as red felt and Mao Liangyan, is a perennial evergreen shrub of the genus Ardisia of the family Myrsinaceae, and is found and introduced for cultivation in a deep mountain aged forest in Dayun county in Jiangxi when wild flower resources are investigated in the mountains of the Ping Ling in Lushan orchard of 1987, the country of origin in south China. The whole tiger tongue is purple red, the leaves are inverted oval, the edges of the leaves are provided with fine saw teeth, the front and back surfaces are sparse in glandular spots, and the red leaves are densely covered with purple thick long villi and shaped like the tongue of a tiger. The red blooming period of the tiger tongue is 6-8 months, the fruit is bright red after being mature, fruits are hung in the whole year, and the fruits and leaves show joyous and auspicious ornamental effects.

On Kunming world gardening fair in 1999, red tiger tongue at Jiangxi exhibitions is the first prize of international indoor leaf-watching plant, and red tiger tongue on the fifth China flower fair (Guangdong Shunde) in 2001 is the first prize, and the red tiger tongue is the first prize to stand at a new base of world-level flowers. In 2019, at Beijing world gardening fair, Rou Zi Qian hong hybrid-Wan Qian hong Rong obtained indoor leaf-watching plant gold prize. The tiger red has good ornamental value and high medicinal value. In recent years, studies on the red tiger tongue are mainly carried out on the cultivation management, chemical component analysis, biological characteristic observation, stress resistance and the like.

Although the germination rate of the red tiger tongue seeds is high, the seedlings are irregular, the seedling emergence process is as long as 2-6 months, the characters of seedlings propagated by the seeds are unstable and easy to change, and excellent single plants are not easy to store and propagate. At present, the regeneration of the red tiger tongue is realized by tissue culture and tends to be mature, but the tissue culture rooting mode of the red tiger tongue is in bottle rooting, and the method needs aseptic operation, has a complex process and a long period. Therefore, a method for rooting the tissue culture seedlings of the Ardisia mamillata Hance outside the bottle, which has the advantages of simplified production process, high rooting rate and high survival rate, is urgently needed.

Disclosure of Invention

The invention aims to provide a method for rooting of Ardisia mamillata Hance tissue culture seedlings outside a bottle, which aims to solve the problems of long rooting production period, low transplanting survival rate and high production cost in the Ardisia mamillata Hance tissue culture seedling bottle.

In order to achieve the purpose, the technical scheme of the invention is as follows: a method for rooting tissue culture seedlings of Ardisia mamillata Hance outside bottles comprises the following steps:

(1) seedling raising plate: 2 seedling raising trays are provided, one seedling raising box is composed of a cover, a hole tray and a water receiving tray 3, the other seedling raising tray is a forest hole tray, and the used seedling raising tray is sprayed with disinfectant solution with the concentration of 0.5-1 g/L, such as carbendazim, chlorothalonil and the like, for disinfection treatment;

(2) preparing moisture: adding tap water into the water receiving tray or the water container in the step (1);

(3) matrix treatment: the rooting medium is vermiculite, perlite and peat; firstly, filling the substrate into the hole tray obtained in the step (1), compacting, and then putting the hole tray into the water receiving tray or the water container obtained in the step (2) to enable the substrate to automatically absorb water and be soaked;

(4) preparing a rooting agent: the rooting agent is IAA (indoleacetic acid), IBA (indolebutyric acid) and NAA (naphthylacetic acid), and is prepared into a solution with the concentration of 50-150 mg/L;

(5) making and processing the cutting: selecting bottle seedlings with good growth vigor of the cluster buds and red stem sections, leaves and fuzz, taking out the bottle seedlings, cutting the cluster buds with the thickness of more than or equal to 1.5mm into stem sections with terminal buds, and cutting off the leaves at the positions of 1-3 cm away from the bases of the stem sections as cuttings; winding the prepared cuttings into bundles by using rubber rings, and soaking 1-3 cm away from the bases of the cuttings in the rooting agent solution in the step (4) for 15-30 min;

(6) the cuttage method comprises the following steps: inserting the cuttings processed in the step (5) into the matrix processed in the step (3), wherein 1 plant is inserted into each hole, the insertion depth is 1-3 cm, and the cuttings are compacted;

(7) the culture conditions are as follows: the culture conditions are 2, one is a tissue culture room, and the other is a greenhouse seedbed;

(8) maintenance management: under the culture condition of the step (7), the cuttings start to root for 15-25 days; opening a vent valve on a cover of the seedling box to acclimate for 3-5 d when the seedling box is cultured in the culture room for 20-25 d, removing the whole seedling box out of the culture room when the whole seedling box is moved to the greenhouse seedbed to be placed for 2-3 d, uncovering and building a sunshade net when the whole seedling box is moved to the greenhouse seedbed for 25-30 d; continuously culturing the seedbed in the greenhouse for about 30d, and opening the plastic films at the two ends of the small arched greenhouse for ventilation;

(9) promoting roots and strengthening seedlings: and (3) culturing seedlings for about 30 days, spraying a poliomyelitis solution with the concentration of 1g/L for 2-3 times at intervals of 5-7 days, and transplanting the seedlings with the root promoting and seedling strengthening time of 15-20 days (culturing for 45-50 days) to a pot.

The principle of the technical scheme is as follows:

(1) matrix: the vermiculite is mica siliceous mineral, has high cation replacement number (CEC), contains more nutrient elements such as potassium, calcium, magnesium and the like, and can be absorbed and utilized by plants to promote growth; the vermiculite is almost aseptic, has plant diseases and insect pests and has strong water absorption capacity after being disinfected at high temperature in the production process, so that the aseptic water and nutrient supply of the red inserts of the tiger tongue during the culture period of the seedling box is ensured;

(2) seedling raising plate: one is a seedling raising box with a cover, and the other is a forest hole tray covered with a film on a small arched shed, so that the water loss and the germ pollution in the hole tray are reduced;

(3) the culture conditions are as follows: one is indoor culture room culture conditions which are relatively consistent with the conditions required by the tissue culture seedling of the red tiger tongue, so that the illumination intensity and time required by the rooting of the cuttings and the temperature condition of the culture are ensured; the other is the outdoor greenhouse seedbed culture condition, the time is selected to be 4-10 months, the tiger tongue is in a red growth period, a film is covered on a small arched greenhouse, the temperature in the film is 25 +/-10 ℃, and the humidity is 90-100%, so that the temperature and humidity conditions during the rooting period of the cuttings are ensured.

The beneficial effects of this technical scheme lie in:

(1) the induction and the domestication are completed simultaneously: the method comprises the steps of inducing the proliferated cluster buds to root outside a bottle, opening a vent valve on a cover for acclimatization for 3-5 d at the 20 th-25 th day of cultivation of a seedling box, and moving out of a cultivation room for cultivation on a greenhouse seedbed; the plastic film at the 2 end of the small arched shed can be opened after the cuttings of the greenhouse seedbed are continuously cultured for about 30 days; the whole rooting process of the cuttings under the 2 culture conditions is the process of simultaneous induction and domestication;

(2) the rooting and seedling time is shortened: according to the whole ex-vitro rooting condition, cuttings are cultured for 15-20 days to start rooting, and the rooting rate reaches 100% after 25-30 days of culture, so that the rooting time is shortened; the root promoting and seedling strengthening culture only needs 15-20 days, so that the seedling time is shortened;

(3) the processes of disinfection and root washing are omitted: through test comparison, the invention determines that vermiculite is used as the optimal ex-bottle rooting matrix, tap water is used for water, and no additional disinfection is needed for the newly opened matrix and the tap water; meanwhile, the invention omits the root washing process in the existing tissue culture seedling in-bottle rooting transplanting method, and avoids root damage caused by root washing and pollution caused by cleaning failure;

(4) is convenient for large-scale production: obtaining the red tiger tongue seedlings with 0 infection rate and 100% rooting in 25-30 days, wherein the average root length of the seedlings after about 30 days of greenhouse seedbed cutting culture is 3.51cm, the root points are 2.92, the average root length of the seedlings after 45-50 days is 4.57cm, the root points are 4.13, the root width is 6-8 cm, and secondary lateral roots are grown to form an intact root group; the whole process is simplified, the rooting rate is high, the infection rate is low, the operation is easy, and the large-scale production is convenient.

Drawings

FIG. 1 is a 12-well seedling raising box without a vent valve (example 1);

FIG. 2 shows the growth effect of test No. 3 cutting for 2 months (example 1) (photographs taken 11 and 16 days 2020);

FIG. 3 shows the effect of the growth of the cuttings of 9 test treatments (example 1) (photographs taken 11/16/2020);

FIG. 4 is a 24-hole seedling raising box with a vent valve (example 2);

FIG. 5 shows a process of loading vermiculite into trays and automatic water absorption (example 2);

FIG. 6 is a well-grown bottle seedling (example 2, example 3);

FIG. 7 shows the production of stem segments with apical buds and cuttings (examples 2 and 3);

FIG. 8 is a cutting into bales and soaked with IAA rooting agent (examples 2, 3);

FIG. 9 shows the insertion of the slip into the plug (example 2);

FIG. 10 is a case where a seedling raising box is placed in a culture chamber for cultivation (example 2);

FIG. 11 is the rooting effect for 15 days of cutting (example 2);

FIG. 12 is the rooting effect (single head) for 25 days of No. E cuttings (example 2);

FIG. 13 is rooting effect (multiheaded) for 30 days of No. e cutting (example 2);

FIG. 14 is a 25-day cutting acclimatization and seedling hardening by opening a vent valve (example 2);

FIG. 15 is a view showing that the nursery box is moved out of the cultivation room to the greenhouse seedbed and the seedling is lifted and hardened on the 3 rd day (example 2);

FIG. 16 is the rooting effect of No. B cuttings for 33 days (example 2) (7 months, 14 days cuttings, 8 months, 17 days photographs);

FIG. 17 is the rooting effect for 53 days of cuttage No. B (example 2) (cuttage on 14 days of 7 months, and photography on 7 days of 9 months);

FIG. 18 is the rooting effect of mixed clone cuttings for 55 days (example 2) (cuttings day 28 month 5, day 3 month 7, removal day 23 month 7);

FIG. 19 shows a flow of a nursery site and a substrate treatment in 50 wells (example 3);

FIG. 20 shows the rooting effect of greenhouse seedbed cuttage for 22 days (example 3) (cuttage in 1 day in 8 months, investigation in 23 days in 8 months)

FIG. 21 shows the rooting effect of greenhouse seedbed cuttings for 30 days (example 3) (7-month 1-day cuttings, 8-month 2-day survey);

FIG. 22 shows the rooting effect (single-headed) of greenhouse seedbed cuttings for 53 days (example 3) (7-month 1-day cuttings, 8-month 23-day survey);

FIG. 23 shows the greenhouse seedbed cutting effect (double heads) for 53 days (example 3) (7 month 1 day cutting, 8 month 23 day survey);

Detailed Description

The method for ex-vitro rooting of the tissue culture seedling of the Ardisia mamillata Hance provided by the invention comprises but is not limited to the following embodiments.

Example 1 screening of substrate and rooting agent

A method for rooting tissue culture seedlings of Ardisia mamillata Hance outside bottles comprises the following steps:

(1) seedling raising box: the seedling raising box consists of a cover, a hole disc and a water receiving disc 3, wherein the length, width and depth of the hole disc are 19.0 cm multiplied by 14.0 cm multiplied by 5.5 cm, the upper hole is 5cm multiplied by 5cm, and the number of the holes of the hole disc is 12 holes (shown in figure 1) of 3 multiplied by 4;

(2) matrix and treatment: in 9/15/2020, the rooting matrix is 1-6 mm granular new vermiculite, perlite and peat (table 1), the matrix is sterilized in an autoclave, then is loaded into a plug tray and compacted, and finally the plug tray is placed into a water receiving tray added with 600-800 ml deionized water to enable the matrix to automatically absorb water and be soaked;

(3) making the cutting: selecting a bottle seedling with good growth vigor of the cluster buds and red stem sections, leaves and fuzz, taking out the cluster buds, cutting the cluster buds into stem sections with terminal buds, and cutting off the leaves at the bases of the stem sections as cuttings;

(4) rooting agent treatment: the rooting agent is IAA (indoleacetic acid), IBA (indolebutyric acid) and NAA (naphthylacetic acid), tap water is used for preparing a solution (table 1) with the concentration of 50-150 mg/L, and 1-3 cm of the base part of the cutting is soaked in the rooting agent solution for 15-30 min;

(5) the cuttage method comprises the following steps: arranging tests (table 2) according to orthogonal test design, processing 12 plants each, performing straight cutting with 1 plant in each hole, inserting the plants with the depth of 1-2 cm, compacting, and covering the seedling raising box cover;

(6) and (3) maintenance management of a culture room: directly placing the cut seedling raising box in a culture room, and managing at normal temperature, wherein the illumination intensity is 2000-2500 Lux, and the illumination time is 12 h.d^-1；

TABLE 1 orthogonal test design Table

Table 2: table of results of orthogonal tests

In 2020, day 11 and 16 (2 months of culture), statistics show that: the substrates of test No. 1, 2 and 3 are vermiculite, the average survival rate and rooting rate reach the maximum value of 72.22%, especially the survival rate and rooting rate of the No. 3 cutting seedling reach 100%, most of the cutting seedlings grow secondary lateral roots, the average rooting point is 2.58, the average root length is 2.48cm (figure 2), and the rooting rate of the cutting seedlings with the substrates of perlite and peat is only 38.89% and 27.78% respectively; for analysis reasons, although the root system of the cutting seedling in peat culture is the best, the cutting pollution is caused to be serious to death due to more germs or easy bacteria infection, and test No. 7 only survives 1 strain and also grows the best 1 strain (figure 3); perlite does not have nutrition, and the cutting slips can form callus tissues after cutting, but can not supply nutrition to plants, so that the root system can not grow; the experiment is only for screening the matrix and the rooting agent, the indoor temperature is 20 +/-3 ℃, and the temperature is low, which is also the reason for longer rooting time.

The rooting rate is 100% (rooting plant number/cutting plant number);

survival rate (number of plants with callus/number of cuttings) 100%;

example 2 improved nursery boxes to continue validation testing in the growth room

(1) seedling raising box: the seedling raising box is composed of a cover, a hole disc and a water receiving disc 3, wherein the length, width and depth of the hole disc are respectively 37 cm multiplied by 23 cm multiplied by 5.8 cm, the specification of the hole is 5cm multiplied by 5cm, and the number of the holes is 24 holes (figure 4) of 6 multiplied by 4;

(2) matrix and treatment: the rooting matrix is 1-3 mm granular vermiculite, the matrix is placed into a plug tray and compacted, the plug tray is placed into a water receiving tray added with about 1300ml of tap water, and the matrix automatically absorbs water and is soaked (figure 5);

(3) making the cutting: selecting a bottle seedling (figure 6) with good growth vigor of the cluster buds and red stem sections, leaves and fuzz, taking out the bottle seedling, cutting the cluster buds with the thickness of more than or equal to 1.5mm into stem sections with 7-8 cm long and terminal buds, and cutting off the leaves at the positions of 1-3 cm away from the base parts of the stem sections as cuttings (figure 7);

(4) rooting agent treatment: the rooting agent is IAA (indoleacetic acid), the concentration is 150mg/L solution, the prepared cuttings are wound into bundles by using rubber rings, and 1-3 cm of the base parts of the cuttings are soaked in the rooting agent solution for 15min (figure 8);

(5) the cuttage method comprises the following steps: 1 plant in each hole is inserted into the hole with the depth of 1-3 cm, compacted and covered with a seedling box cover (figure 9);

(6) culture room conditions: transferring the seedling box to a culture room with illumination intensity of 2000-2500 Lux, illumination time of 12 h.d < -1 > and temperature of 25 +/-3 ℃ for rooting culture (figure 10);

(7) and (3) maintenance management of a culture room: rooting is started after 15-20 d of cuttage (figure 11), multiple cuttings are formed faster than single cuttings along with the growth of root systems and the increasing of the number of roots (figure 12 and figure 13), acclimatization and seedling hardening are carried out after 20-25 d of cultivation by opening a vent valve on a cover (figure 14), and the whole seedling box is moved out of a cultivation room to a greenhouse seedling bed for root promoting and seedling strengthening management after 25-30 d of cuttage (figure 15);

(8) promoting roots and strengthening seedlings: domesticating a seedling box removed from a culture room on a greenhouse seedbed for 2-3 days, uncovering (figure 15), spraying the seedlings with a floral polyhydrous and carbendazim solution with the concentration of 1g/L for 2-3 times at intervals of 5-7 days, and transplanting the seedlings with the root promoting and strong seedlings for 15-20 days (cuttage is carried out for 45-50 days) to a pot; according to investigation, the average root length of 33d of the excellent clone No. B with the Ardisia mamillata Hance is 3.51cm, the average root point is 2.92, the root amplitude is 3-5 cm (figure 16), the average root length of 53d of cutting culture (containing 20d of the root-promoting strong seedling) is 4.29cm, the average root point is 3.58, the root amplitude is 5-7 cm, and a good root ball is formed (figure 17); the hybrid clone of the tiger tongue red is subjected to cuttage culture for 55d (containing 20d of root promoting and strong seedlings), the average root length is 4.57cm, the average root point is 4.13, the root width is 6-8 cm, and seedlings of single head or multiple heads form good root clusters which are obviously superior to the seedlings cultured in the current year (figure 18).

Example 3 verification test with forest plug tray on greenhouse seedbed

(1) seedling raising plate: the seedling raising tray is a forest tree hole tray, and 50 holes (figure 19) with the length, width and depth of 54 cm multiplied by 28 cm multiplied by 9cm, an upper hole of 5cm multiplied by 5cm, a lower hole of 1.5 cm multiplied by 1.5 cm and the number of holes of 5 multiplied by 10 are respectively formed;

(2) matrix and balance: the rooting matrix was identical to example 2, except that the amount of tap water was not controlled, i.e.: 1-3 mm granular vermiculite, filling the substrate into a plug tray, compacting, putting the plug tray into a water container filled with tap water, allowing the substrate to automatically absorb water and soak, and finally putting the plug tray on a greenhouse seedbed (fig. 19);

(3) making the cutting: the slip was made the same as in example 2, except that the length of the slip was not controlled, i.e.: selecting a bottle seedling (figure 6) with good growth vigor of the cluster buds and red stem sections, leaves and fuzz, taking out the bottle seedling, cutting the cluster buds with the thickness of more than or equal to 1.5mm into stem sections with terminal buds, and cutting off the leaves at the positions of 1-3 cm away from the base parts of the stem sections as cuttings (figure 7);

(4) rooting agent treatment: rooting agent treatment was the same as in example 2, i.e.: the rooting agent is IAA (indoleacetic acid), the concentration is 150mg/L solution, the prepared cuttings are wound into bundles by using rubber rings, and 1-3 cm of the base parts of the cuttings are soaked in the rooting agent solution for 15min (figure 8);

(5) the cuttage method comprises the following steps: 1 plant in each hole is inserted with the depth of 1-3 cm, compacted, and sprayed by a sprayer to enable the cuttings to be in close contact with the matrix after cuttage is finished;

(6) the culture conditions are as follows: in a plastic greenhouse, a small arched shed is built on a seedbed, the height of the small arched shed is 60-80 cm, after cuttage is finished, the small arched shed is covered with a film and compacted, and cultivation is carried out at normal temperature and natural illumination;

(7) maintenance management: the hybrid clone with the tiger tongue red begins to root after about 20 days of cutting culture on a greenhouse seedbed (figure 20), when about 30 days, 2-level lateral roots grow out and grow vigorously, the average root length is 3.40cm, the average root point is 4.7, and the root width is 4-5 cm (figure 21);

(8) promoting roots and strengthening seedlings: when the mixed clone of the red tiger tongue is continuously cultured for 30 days, two ends of a plastic film are opened for ventilation, seedlings are sprayed with a poliomyelia solution with the concentration of 1g/L for 2-3 times at intervals of 5-7 d/time, the seedlings with the root promoting and strengthening functions for 15-20 d (cultured for 45-50 d) can be transplanted into a pot, and the average root length of the red tiger tongue seedlings cultured for 53d is 6.5cm, the average root point is 5.5, and the root width is 6-8 cm (shown in figures 22 and 23).

The foregoing is merely an example of the present invention and common general knowledge of known specific structures and features of the embodiments is not described herein in any greater detail. It should be noted that, for those skilled in the art, without departing from the structure of the present invention, several changes and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent.

Claims

1. The ex-vitro rooting method for the tissue culture seedlings of Ardisia mamillata Hance is characterized by comprising the following steps of:

2. The method for ex-vitro rooting of tissue culture seedlings of Ardisia mamillata Hance according to claim 1, which is characterized in that: 2 seedling raising trays are provided in the step (1), one seedling raising box is composed of a cover, a hole tray and a water receiving tray 3, a vent valve is arranged on the cover, the length, width and depth of the hole tray of the seedling raising box are 37 cm multiplied by 23 cm multiplied by 5.8 cm, the hole specification is 5cm multiplied by 5cm, and the number of holes of the hole tray is 24 holes of 6 multiplied by 4; the other is a forest tree hole disk, the length, width and depth of which are 54 cm multiplied by 28 cm multiplied by 9cm, the upper hole of the hole disk is 5cm multiplied by 5cm, the lower hole is 1.5 cm multiplied by 1.5 cm, and the number of holes is 50 holes of 5 multiplied by 10.

3. The method for ex-vitro rooting of tissue culture seedlings of Ardisia mamillata Hance according to claim 1, which is characterized in that: the tap water in the step (2) is household tap water, and can be directly discharged from a tap water pipe without special treatment.

4. The method for ex-vitro rooting of tissue culture seedlings of Ardisia mamillata Hance according to claim 1, which is characterized in that: the rooting matrix in the step (3) is 1-6 mm particles, the matrix is not used or newly unsealed, the matrix is compacted after being palletized, and the matrix is automatically soaked in water.

5. The method for ex-vitro rooting of tissue culture seedlings of Ardisia mamillata Hance according to claim 1, which is characterized in that: and (4) preparing the rooting agent into a solution with the concentration of 50-150 mg/L by using tap water.

6. The method for ex-vitro rooting of tissue culture seedlings of Ardisia mamillata Hance according to claim 1, which is characterized in that: the cutting in the step (5) is to select cluster buds with the thickness of more than or equal to 1.5mm from the bottle seedlings, cut stem sections with terminal buds and cut leaves at the position of 1-3 cm away from the base of the stem sections; and (3) winding the cuttings into a bundle by using a rubber ring, and soaking the cuttings at a position of 1-3 cm away from the base of the cuttings in a rooting solution for 15-30 min.

7. The method for ex-vitro rooting of tissue culture seedlings of Ardisia mamillata Hance according to claim 1, which is characterized in that: the cuttage method in the step (6) is that a hole is punched in a matrix, then the cutting slips are inserted into the matrix, the depth is 1-3 cm, 1 plant is placed in each hole, and the matrix needs to be compacted to enable the cutting slips to be in close contact with the matrix.

8. The method for ex-vitro rooting of tissue culture seedlings of Ardisia mamillata Hance as claimed in claim 1, wherein the culture conditions in step (7) are 2, one is a tissue culture room, namely: after cuttage, a cover of the seedling raising box is covered, a vent valve is closed, the seedling raising box is moved into a tissue culture room, the illumination intensity is 2000-2500 Lux, and the illumination time is 12 h.d^-1At a temperature of 25 +/-3 ℃; the other is a greenhouse seedbed, namely: after cutting, covering a film on a small arched shed, and then culturing at normal temperature and natural light.

9. The method for ex-vitro rooting of tissue culture seedlings of Ardisia mamillata Hance according to claim 1, wherein the cuttings begin to root for 15-25 days under the conditions of maintenance and management in the step (8); opening a vent valve on a cover of the seedling box to acclimate for 3-5 days when the seedling box is cultured in the culture room for 20-25 days, and removing the whole seedling box out of the culture room to a greenhouse seedbed to place the seedling box for 2-3 days, uncovering and building a sunshade net when the seedling box is cultured for 25-30 days; continuously culturing the seedbed in the greenhouse for about 30d, and opening the plastic films at the two ends of the small arched greenhouse for ventilation;

10. the method for rooting outside the tissue culture seedling of Tiger tongue red as claimed in claim 1, wherein the root promoting and seedling strengthening in step (9) is realized by spraying the seedling cultured for about 30 days with a concentration of polyfloride and carbendazim solution of 1g/L for 2-3 times at intervals of 5-7 days, and transplanting the seedling cultured for 15-20 days (45-50 days) into a pot and managing the seedling in the pot.